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. 2014 Jan 30;10(1):e1004125.
doi: 10.1371/journal.pgen.1004125. eCollection 2014 Jan.

Variation in Genome-Wide Levels of Meiotic Recombination Is Established at the Onset of Prophase in Mammalian Males

Free PMC article

Variation in Genome-Wide Levels of Meiotic Recombination Is Established at the Onset of Prophase in Mammalian Males

Brian Baier et al. PLoS Genet. .
Free PMC article


Segregation of chromosomes during the first meiotic division relies on crossovers established during prophase. Although crossovers are strictly regulated so that at least one occurs per chromosome, individual variation in crossover levels is not uncommon. In an analysis of different inbred strains of male mice, we identified among-strain variation in the number of foci for the crossover-associated protein MLH1. We report studies of strains with "low" (CAST/EiJ), "medium" (C3H/HeJ), and "high" (C57BL/6J) genome-wide MLH1 values to define factors responsible for this variation. We utilized immunofluorescence to analyze the number and distribution of proteins that function at different stages in the recombination pathway: RAD51 and DMC1, strand invasion proteins acting shortly after double-strand break (DSB) formation, MSH4, part of the complex stabilizing double Holliday junctions, and the Bloom helicase BLM, thought to have anti-crossover activity. For each protein, we identified strain-specific differences that mirrored the results for MLH1; i.e., CAST/EiJ mice had the lowest values, C3H/HeJ mice intermediate values, and C57BL/6J mice the highest values. This indicates that differences in the numbers of DSBs (as identified by RAD51 and DMC1) are translated into differences in the number of crossovers, suggesting that variation in crossover levels is established by the time of DSB formation. However, DSBs per se are unlikely to be the primary determinant, since allelic variation for the DSB-inducing locus Spo11 resulted in differences in the numbers of DSBs but not the number of MLH1 foci. Instead, chromatin conformation appears to be a more important contributor, since analysis of synaptonemal complex length and DNA loop size also identified consistent strain-specific differences; i.e., crossover frequency increased with synaptonemal complex length and was inversely related to chromatin loop size. This indicates a relationship between recombination and chromatin compaction that may develop as DSBs form or earlier during establishment of the meiotic axis.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Inter-strain variation in mean MLH1 values.
(A) Pachytene cell from B6 male immunostained with antibodies to MLH1 (green) and SYCP3 (red). The number of MLH1 foci per cell were counted and used as a surrogate for meiotic recombination events. (B) The mean number of MLH1 foci per spermatocyte for 5 CAST (n of cells = 105), 7 C3H (n of cells = 209) and six B6 (n of cells = 110) males varied significantly among the three inbred strains. (C, D) For each strain, slides were hybridized with FISH probes for chromosomes 19 and 1 and the positions of MLH1 foci were calculated as a percent of SC length (centromere = 0%; telomere = 100%). (C) On chromosomes 19 with a single MLH1 focus, the focus was typically medially or distally placed; data are based on analysis of 50 chromosomes/strain. (D) On chromosomes 1 with two MLH1 foci, typically one was proximally located and the other distally placed, consistent with positive interference. Data based on analysis of 50 cells/strain.
Figure 2
Figure 2. Inter-strain variation in early- and mid-stage meiotic recombination proteins.
The number and location of foci for proteins acting upstream of MLH1 in the recombination pathway [i.e., (A) RAD51 (B) DMC1 (C) MSH4] or anti-recombination pathway [i.e., (D) BLM] were determined for each of the three strains and inter-strain values compared. For each of the four proteins, mean numbers of foci per cell varied significantly among the three strains. Data for each protein were based on 4–6 animals per strain, and a minimum of 60 cells per strain (see Supplemental Tables 2–5 for data).
Figure 3
Figure 3. Inter-strain variation in SC length and DNA loop size.
(A) Mean autosomal SC lengths varied in direct relationship to MLH1 values; i.e., the strain with the lowest mean MLH1 values (CAST) had the shortest SCs, while the strain with the highest MLH1 values (B6) had the longest SCs. Data are based on analyses of mid-pachytene spermatocytes from 2 CAST (n = 8 cells), 3 C3H (n = 11 cells) and 2 B6 (n = 21 cells) males. (B) DNA loop sizes for individual chromosomes were calculated as the average of the width of the FISH signal at three points along the SC: at the centromere, the midpoint, and the telomere; image shows example for chromosome 12. (C) Chromosomes 1, 12, and 19 were examined and for each, we observed an inverse relationship between strain-specific mean MLH1 values and mean DNA loop sizes.
Figure 4
Figure 4. Spo11+/+ and Spo11+/− animals.
(A) Mice heterozygous for a null allele of Spo11 exhibited a significant decrease in RAD51 foci (a marker of DSBs) by comparison to wildtype littermates. However, (B) the mean number of MLH1 foci (a marker of COs), (C) mean SC lengths and (D) mean DNA loop sizes were not different between the two genotypes.

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